The present invention is concerned with a Newcastle disease virus (NDV) mutant that expresses its V protein at a reduced level, a vaccine comprising the NDV mutant and the use of the NDV mutant for the manufacture of a vaccine to protect birds against ND
Newcastle disease (ND) is one of the devastating diseases of poultry and has substantial economic impact on the poultry industry. NDV is the etiologic agent of this disease and belongs to the family Paramyxoviridae. Newcastle disease is complicated in that different isolates and strains of the virus may induce substantial variation in the severity of the disease. In general, the younger the chicken the more acute and severe the disease. The infection may take place by either inhalation or ingestion of the virus. The infectious form of the virus spreads from one bird to another.
In order to reduce the economic losses due to ND in the poultry industry, vaccination of chickens, particularly those raised for commercial consumption, is carried out throughout the world on a routine basis. Examples of live (lentogenic) NDV vaccine strains commonly used are the V4, Hitchner B 1, F and La Sota strain. However, these vaccine strains still cause light to moderate vaccination reactions, in particular in the respiratory tract upon primary vaccination of young birds.
Mild NDV vaccine strains have been developed which do not cause (respiratory) vaccination reactions upon administration to young birds: U.S. Pat. No. 5,250,298 (University of Delaware) discloses a live, cold-adapted temperature-sensitive mutant of the Hitchner B1 strain, designated as CaTs. U.S. Pat. No. 5,149,530 (Duphar Int. Res. B.V.) describes a NDV strain, designated as NDW, which is a mutant derived from the Ulster 2C strain. U.S. Pat. No. 5,750,111 (Akzo Nobel N.V.) discloses a mild vaccine strain, designated as the C2 strain, which does not induce adverse reactions in one-day-old chicks.
Currently available NDV vaccines can only be administered to hatched chickens through drinking water, aerosol, eye drops or by parenteral routes. These methods of applications have some disadvantages, most importantly expensive because of the labour needed for their application. Recently, the use of vaccines, such as herpesvirus of turkey and infectious bursal disease virus vaccines as embryo vaccines (Sharma and Burmester, Avian Diseases 26, 134-149, 1982 and Sharma, Avian Diseases 29, 1155-1169, 1985) has proved to be effective and economical. Moreover, embryo vaccination was found to be advantageous due to early age of resistance to the specific disease and administration of a uniform dose of vaccine into each egg using semiautomatic machines with multiple injection heads.
It should be noted that many vaccines used conventionally for post-hatch vaccination of birds cannot be used for in ovo vaccination. Late stage embryos are highly susceptible to infection with most vaccine viruses examined, including those vaccine viruses which can safely be used in one-day-old hatched chicks. Consequently, conventional vaccines must be modified for in ovo use.
Currently, there is no suitable commercially available ND vaccine that can be applied in ovo, mainly due to high level of embryo mortality associated even with two of the mildest commercially available NDV vaccine strains: NDW and C2. U.S. Pat. No. 5,427,791 (Regents of the University of Minnesota) discloses the use of chemical mutagenic agents to produce NDV mutants of the Hitchner BI strain that are non-pathogenic for late stage embryos. Chemical treatment of the BI strain with ethyl methanine sulfonate (EMS) resulted in the mutant virus NDV-B1-EMS which could be safely administered to chicken eggs at embryonation day 18. However, such mutagenic process leads to the introduction of random mutations in the genome of the virus in an uncontrolled, non-reproducible way. Such random mutations may influence properties of the virus other than those associated with the safety in ovo, such as properties of the virus related to the immunogenicity. Moreover, disadvantageously, each egg passage step of this strain must be carried out in the presence of the mutagenic agent EMS because of the property of the mutant to revert back to the parent B1 strain which is not safe for embryos.
Recently, genetic modification of non-segmented negative stranded RNA viruses has become possible by the development of a process referred to as xe2x80x9creverse geneticsxe2x80x9d (reviewed in Conzelmann, J. Gen. Virology 77, 381-389, 1996; Conzelmann, Annu. Rev. Genet. 32, 123-162, 1998 and Palese et al., Proc. Natl. Acad. Sci. 93, 11354-11358, 1996). The established reverse genetics system that enable controlled genetic manipulation of negative strand RNA viruses has potential applications for the development of novel vaccine strains.
NDV is a member of the family Paramyxoviridae and its negative-strand RNA virus genome contains six genes encoding six major structural proteins (3xe2x80x2NP-P-M-F-HN-L 5xe2x80x2). A general feature of paramyxoviruses, however, is the presence of additional structural or non-structural viral proteins resulting from the use of alternative reading frames and RNA editing of their P gene (reviewed by Kolakofsky et al., J. Virology 72, 891-899, 1998). Like other paramyxoviruses, NDV is also found to edit its P gene by inserting one or two G residues at the editing locus (UUUUUCCC). The three mRNAs encode the P protein (unedited), the V ORF (with +1 frame-shift) and the W ORF (with +2 frame-shift) (Steward et al., J. Gen. Virology 74, 2539-2547, 1993). Translation of the P, V and W specific mRNAs result in the expression of three proteins which have the same N-terminal halve but which differ in their C-terminal halves as a result of the use of different reading frames down-stream of the editing locus.
Peeters et al. (J. Virology 73, 5001-5009, 1999) and Rxc3x6mer-Oberdxc3x6rfer et al. (J. Gen. Virol. 80, 2987-2995, 1999) described the generation of infectious NDV entirely from cloned cDNA by the reverse genetics system. It is shown in Peeters et al (1999) that the virulence of a NDV vaccine strain can be increased dramatically by modifying the amino acid sequence at the cleavage site of the F0 protein. It is also suggested that that elimination of expression of the V protein of NDV may result in an attenuated phenotype in birds (Peeters et al., 1999, supra).
It is an object of the present invention to identify a NDV mutant which can be used for the manufacture of a vaccine for the protection of birds against ND which can be administered not only to young birds after hatch, but which can also be administered safely in ovo.
A new NDV mutant has been identified herein that displays not only mild, attenuated properties for young hatched chickens similar to those displayed by the commercially available mild NDW and C2 vaccine strains, but which in contrast to the NDW and C2 vaccine strains, can also safely be used for embryo vaccination.
The invention provides a NDV mutant that expresses its V protein at a reduced level (NDV Vxe2x88x92), characterised in that the mutant is phenotypically V protein positive and wherein xe2x89xa66% of its P-gene derived mRNAs in infected cells encode V ORF.
It has been found that a NDV mutant as defined above causes significantly less embryo mortality, even if administered to embryos at 11 days of age. This is in contrast to the parent lentogenic vaccine strain from which the mutant is derived. This vaccine strain kills all the embryos before they hatch. Additionally, it was found that a NDV Vxe2x88x92 mutant does not affect hatchability of the eggs, particularly of embryonated commercial chicken eggs and that chickens hatched from embryo vaccinated eggs were protected against virulent NDV challenge. These unexpected combined properties of a NDV Vxe2x88x92 mutant make such a mutant especially suitable for the manufacture of a vaccine for in ovo administration.
Surprisingly, it has been found that NDV mutants that are not able to express V protein generated by reverse genetics techniques can not be rescued after passage of transfection supernatants into embryonated chicken eggs. The complete elimination of V protein expression of a NDV mutant does not result in infectious virus particles and, hence, should be prevented.
Therefore, a NDV mutant according to the invention is phenotypical positive but immunological tests demonstrate that the level of V protein produced in an infected cell is reduced when compared with a cell infected with the parent NDV. The presence or absence (the phenotype) and relative level of V protein expression in an infected host cell can be determined in an immune fluorescence test (IFT) or immunoblotting using a specific V protein antiserum raised against the C-terminus of the V protein as described herein.
A NDV Vxe2x88x92 mutant according to the invention clearly displays defective P-gene mRNA editing. Compared to cells infected with the parent NDV in which the P-gene derived mRNAs encoding V ORF (and W ORF) are generally present at a frequency of approximately 30% (and 2%), a NDV Vxe2x88x92 mutant according to the invention edits its P gene at a frequency of only xe2x89xa66%.
The relative occurrence of the P-gene derived mRNA populations can be determined as described in Example 1. In this respect, the number of clones used for the determination of the P-gene mRNA editing frequency should be at least 100, preferably between 100 and 500. For a phenotypical V protein positive NDV Vxe2x88x92 mutant, V ORF editing is higher than 0%.
Preferably, the phenotypical V protein positive NDV Vxe2x88x92 mutant is a mutant wherein xe2x89xa63% and more preferably xe2x89xa61% of its P-gene derived mRNAs in infected cells encode V ORF.
Alternatively, a NDV Vxe2x88x92 according to the invention can also be defined by means of the level of its V ORF editing frequency: the NDV Vxe2x88x92 mutant according to the invention displays a percentage V ORF editing frequency (e.f.) of 0  less than e.f. xe2x89xa66, preferably 0  less than e.f. xe2x89xa63, more preferably 0  less than e.f xe2x89xa61.
The NDV Vxe2x88x92 mutant can be used for the manufacture of a ND vaccine for in ovo administration according to standard methods as commonly used for the preparation of conventional live ND vaccines.
Briefly, a susceptible substrate is inoculated with a NDV Vxe2x88x92 mutant and propagated until the virus replicated to a desired titre after which NDV containing material is harvested. Subsequently, the harvested material is formulated into a pharmaceutical preparation with immunising properties.
Every substrate which is able to support the replication of ND viruses can be used in the present invention, including primary (avian) cell cultures, such as chicken embryo fibroblast cells (CEF) or chicken kidney cells (CK), or mammalian cell lines such as the VERO cell line or baby hamster kidney (BHK) cell line.
Particularly suited substrates on which the NDV Vxe2x88x92 mutant can be propagated are SPF embryonated eggs. Embryonated eggs can be inoculated with, for example 0.2 ml NDV containing allantoic fluid comprising at least 102.0 EID50 per egg. Preferably, 9-12 day-old embryonated eggs are inoculated with about 105.0 EID50 and subsequently incubated at 37xc2x0 C. for 2-4 days. After 2-4 days the ND virus product can be harvested preferably by collecting the allantoic fluid. The fluid can be centrifuged thereafter for 10 min. at 2500 g followed by filtering the supernatant through a filter (100 xcexcm).
The vaccine to be used for the in ovo administration comprises the live ND virus mutant and a pharmaceutically acceptable carrier or diluent customary used for such compositions. The vaccine can be prepared and marketed in the form of a suspension or in a lyophilised form. Carriers include stabilisers, preservatives and buffers. Suitable stabilisers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline) and polyols (such as glycerol).
The vaccine comprising the NDV Vxe2x88x92 mutant can be injected into embryonated eggs according to conventional in ovo vaccination methods. Usually, the vaccine is injected into embryonated eggs during late stages of the embryonation, generally during the final quarter of the incubation period (day 15-21), preferably at day 18 of the incubation period.
The mechanism of injection of the incubated eggs is not particularly critical provided that it does not unduly damage tissue and organs of the embryo. For example, a small hole is pierced with a needle (11xc2xd inch, about 22 gauge) attached to syringe in the large end of the shell and the vaccine is injected below the inner shell membrane and the chorioallantoic membrane. Subsequently, the vaccinated embryonated eggs are transferred to an incubator to hatch (U.S. Pat. No. 4,458,630, 5,427,791, WO 98/56413 and WO 95/35121). Preferably, the whole embryo vaccination process is carried out using automated vaccination systems, such as the commercially available Inovoject(copyright).
The NDV Vxe2x88x92 mutant used for the manufacture of the ND vaccine for in ovo administration can be prepared according to the established reverse genetics method which has already been used for the genetic modification of many non-segmented, negative stranded RNA viruses (for reviews see above). Additionally, such a method has also been disclosed for NDV by Peeters et al. (1999, supra) and Rxc3x6mer-Oberdxc3x6rfer et al. (1999, supra).
Typically, first, a full length cDNA clone of the NDV genome is assembled (from overlapping cDNA fragments) and cloned into a transcription plasmid between a (T7) RNA polymerase promoter and an autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that express a (T7) RNA polymerase results in the synthesis of antigenome NDV RNA. Simultaneous expression from co-transfected plasmids of the viral proteins that are required for virus replication and transcription (NP, P and L proteins) result in the generation of infectious virus from cloned cDNA. The nucleotide sequences of all NDV genes are known. The nucleotide sequence of the P gene have been described by Ishida et al., NAR 14, 6551-6564, 1986; McGinnes et al., Virology 164, 256-264, 1988; Daskalakis et al., NAR 20, 616, 1992 and Steward et al., J. Gen. Virology 74, 2539-2547, 1993). The nucleotide sequence of the complete NDV genome has also been reported by several research groups (de Leeuw et al., J. Gen. Virology 80, 131-136, 1999, GenBank accession no. AF077761; Krishnamurthy et al., J. Gen Virology 79, 2419-2424, 1998, Phillips et al., Arch. Virol. 143, 1993-2002, 1998, EMBL accession no. AJ225127, AJ225128 and AJ225129 and Romer-Oberdorfer et al., J. Gen. Virol. 80, 2987-2995, 1999; EMBL accession no. Y18898). The length of the complete NDV genome is 15,186 nucleotides including the 3xe2x80x2- and 5xe2x80x2- terminal ends.
The P gene is located on the NDV genome at nucleotides 1804-3254 (NDV strain Clone 30(copyright) numbering as used by Romer-Oberdorfer et al., EMBL accession no. Y18898; this numbering will be used herein to identify positions on the NDV genome). The open reading frame (ORF) encoding the P protein is located at nucleotides 1887-3074. The P gene mRNA editing locus UUU UUC CC (genome RNA-sense) to be mutated resulting in a NDV Vxe2x88x92 mutant is located at position 2280-2287. The end of the ORFs encoding the P, V and W protein are at positions 3074 (TAA), 2605 (TAA) and 2424 (TGA), respectively. The P protein is 395 amino acids long and the N-terminal halve of the P protein which is identical to the N-terminal halve of the V protein (and W protein) extends from amino acid 1-135. The C-terminal halves of the P and V proteins, i.e. the fragments of the P and V protein that share no sequence homology, extend from amino acid 136-395 and 136-239, respectively. Because of the (+1) frame-shift at the end of the editing locus during transcription, the C-terminal halves of the P- and V protein do not show any similarity.
Preferably, the present invention provides a NDV Vxe2x88x92 mutant that expresses its V protein at a reduced level as a result of a mutation in the editing locus UUU UUC CC. The disturbance of this highly specific sequence results in a reduction of the insertion frequency of non-templated G residue(s) at the editing site during transcription and, consequently, in a reduction of V (and W) protein expression.
A mutation is understood to be a change of the genetic information in the editing locus of the P gene of a parent NDV strain which is able to express a V protein. The mutation is, in particular, a nucleic acid substitution.
In particular, a nucleic acid substitution is introduced in one of the codons of the editing locus which results in a silent mutation, i.e. a mutation which alters the codon but not the amino acid encoded by that codon. Such a mutation guarantees that the ORF of the P gene still expresses a functional P protein. Examples of silent mutations at the conserved editing site including a mutation at position 3 (UUC UUC CC) or position 6 (UUU UUU CC) or a combination of both mutations (UUC UUU CC) are within the scope of this invention.
As demonstrated in Example 1, substitutions of 3 or more nucleotides and deletions of nucleotides result in NDV mutants that are not able to express V protein and can not be rescued from the transfection supernatants. Therefore, the substitution in the editing locus of a NDV mutant according to the invention comprises 1 or 2 nucleotides. Furthermore, it is demonstrated in Example 1 that mutations introduced at position 1-5 involving 1 or 2 nucleotides of the editing locus advantageously result in a NDV mutant that can be rescued from transfection supernatants and displays a reduced V expression of approximately 20-fold lower than that of the parent virus. Moreover, all the mutants are considerably attenuated in pathogenicity for chicken embryos. In view of these findings, a NDV mutant as described above having 1 or 2 mutations at position 1-5 of the editing locus, preferably at postion 3 or 4 is specifically contemplated.
A very advantageous example of a NDV Vxe2x88x92 mutant according to the invention having a mutation at position 3 of the editing locus comprises the nucleotide sequence UUC UUC CC at the editing locus. Although the first codon of the editing locus of this mutant is changed, the amino acid encoded by this codon remains the same (a lysine residue). This NDV Vxe2x88x92 mutant dramatically reduces P gene mRNA editing as demonstrated by the reduction (to xe2x89xa66%) of V (and W to undetectable levels) ORF mRNAs. NDV Vxe2x88x92 mutants demonstrating a similar reduction of V ORF editing can also be obtained by other single nucleotide substitutions in the editing locus. In such a NDV Vxe2x88x92 mutant a U residue of the editing locus is substituted by C, G or A residue, preferable by a C residue, or a C residue of the editing locus is substituted by a U, G or A residue, preferably by a U residue.
Advantageous examples of such NDV Vxe2x88x92 mutants are NDV mutants comprising the nucleotide sequence UCUUUCCC, UUUGUCCC and UUUUCCCC at the editing locus.
A typical example of a NDV mutant according to the invention having 2 mutations at the editing locus comprises the nucleotide sequence GCUUUCCC.
The desired mutations can be introduced into the NDV genome by means of methods generally known in the art for this purpose. In particular, the mutations are introduced by means of site-directed mutagenesis. Such a method is described herein, but is also generally used in the art (Peeters et al., 1999, supra; Current Protocols in Molecular Biology, eds.: F. M. Ausubel et al., Wiley N.Y., 1995 edition, pages 8.5.1.-8.5.9.and Kunkel et al., Methods in Enzymology Vol. 154, 376-382, 1987).
A particular preferred NDV Vxe2x88x92 mutant to be used according to the present invention is a NDV mutant as described above which comprises additional attenuating mutations. Such NDV mutants can be derived from any ND vaccine strain. Examples of such suitable NDV vaccine strains present in commercially available ND vaccines are: Clone-30(copyright), La Sota, Hitchner B1, NDW, C2 and AV4, Clone-30(copyright) being the preferred vaccine strain.
In another aspect, the present invention provides a live vaccine against ND in birds suitable for in ovo administration, characterised in that the vaccine comprises a NDV Vxe2x88x92 mutant as described above, together with a pharmaceutical acceptable carrier.
Typically, such a vaccine comprises doses of 100 xcexcl or less, preferably 50 xcexcl, per egg. The administration of the in ovo vaccine in such small dosages volumes improves the hatchability of the vaccinated embryos.
In a further embodiment of the present invention a live combination vaccines is provided which, in addition to the NDV Vxe2x88x92 mutant described above, comprises an embryo-safe vaccine strain of another avian pathogen. The combined administration of more than one vaccine strain is advantageous for economical reasons, because it requires fewer vaccine inoculations in the egg. Moreover, the fewer a needle is introduced into an egg, the less risk of contaminating the eggs exists.
With an embryo-safe vaccine strain is meant a live vaccine strain which, if inoculated into SPF eggs at embryonation day 18, results in the hatchability of the eggs of at least 70%, preferably at least 90%. In particular, the combination vaccine additionally comprises one or more embryo-safe vaccine strains of Marek""s disease virus (MDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), fowl adenovirus (FAV), turkey rhinotracheitis virus (TRTV) or reovirus. Examples of such embryo-safe vaccine strains are the MDV vaccines Ovovac(copyright)-HVT and Ovovac(copyright)-SB1, the IBDV vaccines Bursamune(copyright) and Bursaplex(copyright).
It will be clear that because of the advantageous attenuated properties of the NDV V mutant as shown herein, the live vaccine according to the present invention can also be administered to birds after hatch in a similar way as the live ND vaccines which are routinely used to prevent ND in commercial flocks.
In still a further embodiment of the present invention a vector vaccine is provided which can be used not only for the preparation of a vaccine against infection by a specific NDV, but also against other poultry infectious diseases. For example, a vector vaccine based on a NDV Vxe2x88x92 mutant as described above offers the possibility to immunise against other avian pathogens by the expression of antigens of these avian pathogens in infected cells of the immunised host. Such a NDV vector can be obtained by inserting a heterologous nucleic acid sequence encoding a polypeptide heterologous into a non-translated region of the NDV Vxe2x88x92 mutant. Non-translated region suitable for this purpose are located between the genomic promoter and the start of the NP gene, and at the NP/P, P/M, M/F, F/HN and HN/L gene junctions. The heterologous nucleic acid sequence may encode an antigen of an avian pathogen such as infectious bursal disease virus, infectious bronchitis virus, Marek""s disease virus, avian encephalomyelitis virus, avian reovirus, avian influenza virus, chicken anaemia virus, Salmonella spp., E.coli, and Eimeria spp.
The NDV Vxe2x88x92 mutant described above also offers the possibility to prepare an inactivated vaccine with advantageous properties for post-hatch administration. An important advantage of such an inactivated vaccine is the high level of protective antibodies of long duration that can be achieved as a result of the high antigenic mass produced by the NDV Vxe2x88x92 mutant upon propagation in embryonated eggs or cell culture.
The aim of inactivation of the ND viruses harvested after the propagation step is to eliminate reproduction of the viruses. In general, this can be achieved by chemical or physical means. Chemical inactivation can be effected by treating the viruses with, for example, enzymes, formaldehyde, xcex2-propiolactone, ethylene-imine or a derivative thereof. If necessary, the inactivating compound is neutralised afterwards. Material inactivated with formaldehyde can, for example, be neutralised with thiosulphate or sodium metabisulfite. Physical inactivation can preferably be carried out by subjecting the viruses to energy-rich radiation, such as UV light, X-radiation or xcex3-radiation. If desired, the pH can be brought back to a value of about 7 after treatment.
A vaccine containing the inactivated ND virus can, for example, comprise one or more of the above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose.
Preferably, an inactivated vaccine according to the invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminium hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol F(copyright) or Marcol 52(copyright) or a vegetable oil such as vitamin E acetate, and saponins.
The vaccine according to the invention comprises an effective dosage of the NDV Vxe2x88x92 mutant as the active component, i.e. an amount of immunising NDV material that will induce immunity in the vaccinated birds against challenge by a virulent ND virus. Immunity is defined herein as the induction of a significant higher level of protection in a population of birds after vaccination compared to an unvaccinated group.
Typically, the live vaccine according to the invention can be administered in a dose of 103.0-108.0 embryo infectious dose50 (EID50) per egg or bird, preferably in a dose ranging from 104.0-107.0 EID50, in particular 105.0-107.0 EID50.
Inactivated vaccines may contain the antigenic equivalent of 104.0-109.0 EID50 per animal, preferably between 106.0-108.0 EID50 per animal.
Inactivated vaccines are administered parenterally, e.g. intramuscularly or subcutaneously.
Although, the vaccine according to the present invention may be used effectively in chickens, also other poultry such as turkeys, guinea fowl and partridges may be successfully vaccinated with the vaccine.
NDVs have also been described as therapeutic agents in humans, particularly in the treatment of human cancer (Lorence et al., J.Natl Cancer Inst. 80, 1305-1312, 1988; Murray et al., Cancer 52, 856-862, 1983; Reichard et al., J.Surg.Res. 52, 448-453, 1992). Since NDV causes conjunctivitis in humans, a highly attenuated NDV strain is desired for therapy purposes. Therefore, in view of the advantageous properties of the NDV Vxe2x88x92 mutant described above, the NDV mutant (if desired, comprising a foreign gene encoding a therapeutic or prophylactic protein) may be used as a therapeutic agent in humans, e.g. in the control of human or animal cancer and AIDS.